Autophagy-associated Protein Levels Research Articles

Electroacupuncture alleviates sciatic nerve injury and inhibits autophagy in rats.

Sciatic nerve injury is a common form of peripheral nerve injury (PNI). It has been suggested that electroacupuncture (EA) stimulation at GB30 and ST36 can improve nerve dysfunction post-PNI. Autophagy is an important factor in the regeneration of sciatic nerves and recovery of motor function. Therefore, we investigated the biological effects of EA and examined whether these were mediated by autophagy in sciatic nerve injury. Mechanical clamping of the sciatic nerve in Sprague-Dawley rats was performed to establish an experimental model of sciatic nerve injury. EA stimulation was administered once daily for 15 min for seven consecutive days beginning 1 week after successful modeling. The recovery of sciatic nerve function was examined via the sciatic functional index (SFI) test. Morphometric analysis was conducted by staining nerve samples with toluidine blue. Autophagy-associated protein levels were measured via Western blotting. EA stimulation at GB30 and ST36 significantly increased the number of myelinated fibers, axonal and fiber diameters, and the thickness of the myelin sheath in our rat model of sciatic nerve injury. In addition, EA stimulation greatly facilitated nerve regeneration following sciatic nerve injury. Moreover, sciatic nerve injury-induced autophagy was inhibited by EA stimulation. EA facilitates recovery of injured sciatic nerves and inhibits autophagy in a rat model.

Regulation of platelet activation and thrombus formation in acute non-ST segment elevation myocardial infarction: Role of Beclin1.

This study aims to investigate the mechanism of platelet activation-induced thrombosis in patients with acute non-ST segment elevation myocardial infarction (NSTEMI) by detecting the expression of autophagy-associated proteins in platelets of patients with NSTEMI. A prospective study was conducted on 121 patients with NSTEMI who underwent emergency coronary angiography and optical coherence tomography. The participants were divided into two groups: the ST segment un-offset group (n = 64) and the ST segment depression group (n = 57). We selected a control group of 60 patients without AMI during the same period. The levels of autophagy-associated proteins and the expression of autophagy-associated proteins in platelets were measured using immunofluorescence staining and Western blot. In NSTEMI, the prevalence of red thrombus was higher in the ST segment un-offset myocardial infarction (STUMI) group, whereas white thrombus was more common in the ST segment depression myocardial infarction (STDMI) group. Furthermore, the platelet aggregation rate was significantly higher in the white thrombus group compared with the red thrombus group. Compared with the control group, the autophagy-related protein expression decreased, and the expression of αIIbβ3 increased in NSTEMI. The overexpression of Beclin1 could activate platelet autophagy and inhibit the expression of αIIbβ3. The results suggested that the increase in platelet aggregation rate in patients with NSTEMI may be potentially related to the change in autophagy. And the overexpression of Beclin1 could reduce the platelet aggregation rate by activating platelet autophagy. Our findings demonstrated that Beclin1 could be a potential therapeutic target for inhibiting platelet aggregation in NSTEMI.

Metformin Ameliorates Postoperative Cognitive Dysfunction through Regulation of the AMPK/SIRT1 Pathway.

Postoperative cognitive dysfunction (POCD) is a common postoperative complication in elderly patients. The purpose of this study was to investigate the mechanism through which metformin improves postoperative cognitive function. In the in vivo experiment, 18-month-old Sprague-Dawley (SD) rats were randomly divided into four groups (n = 12 in each group): the control, metformin, operation, and operation plus metformin groups. The animals were pretreated with metformin by gavage once daily for two weeks. The Morris water maze (MWM) was used to measure cognitive ability. In the in vitro experiment, BV2 cells were divided into five groups: the control, metformin, lipopolysaccharide (LPS), LPS plus metformin, and LPS plus metformin plus compound C groups. We stimulated microglia with LPS (500 ng/mL). Immunofluorescence and Western blotting were used to assess ROS (reactive oxygen species) levels, autophagy-associated protein levels and adenosine monophosphate-activated protein kinase (AMPK)/regulator factor 2-related enzyme 1 (SIRT1) signaling pathway activity in the rat cortex and microglial cells. In the MWM test, the metformin-pretreated rats spent a higher proportion of time in the target quadrant. Immunofluorescence showed that the fluorescence intensity of LC3 in the cortex was increased in rats pretreated with metformin. Western blotting indicated that metformin upregulated the expression of autophagy-related and AMPK/SIRT1 signaling pathway-related proteins in the cortex after surgery. By activating the AMPK/SIRT1 signaling pathway in vitro, metformin reduced microglial activation and oxidative stress and promoted autophagy. Through the AMPK/SIRT1 pathway, metformin can boost autophagy and reduce oxidative stress in cortical microglia in older rats, in turn improving postoperative cognitive function.

High Autophagy Patterns in Swelling Platelets During Apheresis Platelet Storage.

Platelets undergo remarkable morphological changes during storage. Platelets change into different sizes and densities and differ in their biochemistry and functions. However, the correlation between structural heterogeneity and platelet autophagy is largely unknown. The aim of this study was to investigate the autophagy process in vitro, such as routine storage of platelets, and explore the role of reactive oxygen species (ROS) involved in the regulation of platelet autophagy. The ROS and autophagy levels of platelet concentrates from apheresis platelets were evaluated through flow cytometry. The expression levels of autophagy-associated proteins (LC3I, LC3II, Beclin1, Parkin, and PINK1) were measured via Western blot. All biomarkers were dynamically monitored for seven days. Moreover, the morphological characteristics of platelet morphology during storage were analyzed through transmission electron microscopy (TEM). Flow cytometry showed that the levels of total cell ROS and mitochondria ROS increased in the stored platelets. Together with the increase in mitochondrial ROS, the autophagy signal LC3 in the platelets was strongly amplified. The number of swollen platelets (large platelets) considerably increased, and that of autophagy signal LC3 was remarkably higher than that of the normal platelets. Western blot revealed that the expression levels of Beclin1 and LC3 II/LC3 I ratio were enhanced, whereas those of Parkin and PINK1 almost did not change during the seven days of storage. The existence of autophagosomes or autophagolysosomes in the platelets at the middle stage of platelet storage was observed via TEM. Our data demonstrated that the subpopulation of large (swollen) platelets exhibited different autophagy patterns. Furthermore, increased platelet autophagy was associated with mitochondrial ROS.These preliminary results suggest that swelling platelets have a higher autophagy pattern than normal platelets during storage.

Role of Autophagy and Apoptosis in Aluminum Exposure-Induced Liver Injury in Rats.

Aluminum (Al) exposure can lead to different degrees of damage to various organ systems of the body. It has been previously revealed that Al exposure can damage the liver, causing liver dysfunction. However, the specific mechanism remains unclear. This research aims to uncover the damaging effect of Al exposure on rat liver and to demonstrate the role of autophagy and apoptosis in this effect. Thirty-two Wistar rats were randomly divided into the control group (C group), low-dose Al exposure group (L group), middle-dose Al exposure group (M group), and high-dose Al exposure group (H group) (n = 8). The rats, respectively, received intraperitoneal injections of 0, 5, 10, and 20mg/kg·day AlCl3 solution for 4weeks (5 times/week). After the experiment, changes in the ultrastructure and autolysosome in rat liver were observed; the liver function, apoptosis rate, as well as levels of apoptosis-associated proteins and autophagy-associated proteins were detected. The results indicated that Al exposure damaged rat liver function and structure and resulted in an increase in autolysosomes. TUNEL staining revealed an elevated number of apoptotic hepatocytes after Al exposure. Moreover, we found from Western blotting that the levels of autophagy-associated proteins Beclin1 and LC3-II were increased; apoptotic protein Caspase-3 level was elevated and the Bcl-2/Bax ratio was reduced. Our research suggested that Al exposure can lead to high autophagy and apoptosis levels of rat hepatocytes, accompanied by hepatocyte injury and impaired liver function. This study shows that autophagy and apoptosis pathways participate in Al toxication-induced hepatocyte injury.

NRF2 activation by 2-methoxycinnamaldehyde attenuates inflammatory responses in macrophages via enhancing autophagy flux

A well-controlled inflammatory response is crucial for the recovery from injury and maintenance of tissue homeostasis. The anti-inflammatory response of 2-methoxycinnamaldehyde (2-MCA), a natural compound derived from cinnamon, has been studied; however, the underlying mechanism on macrophage has not been fully elucidated. In this study, LPS-stimulated production of TNF-α and NO was reduced by 2-MCA in macrophages. 2-MCA significantly activated the NRF2 pathway, and expression levels of autophagy-associated proteins in macrophages, including LC3 and P62, were enhanced via NRF2 activation regardless of LPS treatment, suggesting the occurrence of 2-MCA-mediated autophagy. Moreover, evaluation of autophagy flux using luciferase-conjugated LC3 revealed that incremental LC3 and P62 levels are coupled to enhanced autophagy flux. Finally, reduced expression levels of TNF-α and NOS2 by 2-MCA were reversed by autophagy inhibitors, such as bafilomycin A1 and NH4Cl, in LPS-stimulated macrophages. In conclusion, 2-MCA enhances autophagy flux in macrophages via NRF2 activation and consequently reduces LPS-induced inflammation.

Protective effect of ghrelin on intestinal I/R injury in rats.

This study aimed to investigate whether ghrelin affected the autophagy and inflammatory response of intestinal intraepithelial lymphocytes (IELs) by regulating the NOD2/Beclin-1 pathway in an intestinal ischemia–reperfusion (I/R) injury model. Twenty hours after implementing the intestinal I/R injury rat model, the small intestine and both lungs were collected for histological analysis. The morphological changes in the intestinal mucosa epithelium and lung tissues were evaluated using hematoxylin-eosin staining. The activity of autophagic vacuoles and organ injury were evaluated using electron microscopy. The cytokine levels (IL-10 and TNF-α) in IEL cells and lung tissue were determined using enzyme-linked immunosorbent assay. RT-qPCR and western blot assays were conducted to check the NOD2, Beclin-1, and ATG16 levels. Ghrelin relieved the I/R-induced destruction of the intestinal mucosa epithelium and lung tissues. Moreover, ghrelin enhanced autophagy in the intestinal epithelium and lungs of I/R rats. In addition, the levels of autophagy-associated proteins (Beclin-1, ATG16, and NOD2) were higher in the ghrelin treatment group than in rats with I/R. Ghrelin reduced significantly the IL-10 and TNF-α levels. However, these changes were reversed by the NOD2 antagonist. In conclusion, ghrelin may relieve I/R-induced acute intestinal mucosal damage, autophagy disorder, and inflammatory response in IELs by regulating the NOD2/Beclin-1 pathway.

Hydrogen therapy after resuscitation improves myocardial injury involving inhibition of autophagy in an asphyxial rat model of cardiac arrest

Hydrogen (H2) therapy is a therapeutic strategy using molecular H2. Due to its ability to regulate cell homeostasis, H2 therapy has exhibited marked therapeutic effects on a number of oxidative stress-associated diseases. The present study investigated the effectiveness of H2 therapy in protecting against myocardial injury in a rat model of asphyxial cardiac arrest and cardiopulmonary resuscitation. Rats underwent 10-min asphyxia-induced cardiac arrest (CA) and cardiopulmonary resuscitation (CPR), and were randomly divided into control and H2 therapy groups. After resuscitation, the H2 therapy group was administered room air mixed with 2% H2 gas for respiration. During CA/CPR, the arterial pressure and heart rate were measured every minute. Survival rate, cardiac function, myocardial injury biomarkers creatine kinase-MB and cardiac troponin-T, and histopathological changes were evaluated to determine the protective effects of H2 therapy in CA/CPR. Immunohistochemistry and western blot analysis were used to determine the expression levels of autophagy-associated proteins. In vitro, H9C2 cells were subjected to hypoxia/reoxygenation and H2-rich medium was used in H2 treatment groups. Western blotting and immunofluorescence were used to observe the expression levels of autophagy-associated proteins. Moreover, an adenovirus-monomeric red fluorescent protein-green fluorescent protein-LC3 construct was used to explore the dynamics of autophagy in the H9C2 cells. The results showed that H2 therapy significantly improved post-resuscitation survival and cardiac function. H2 therapy also improved mitochondrial mass and decreased autophagosome numbers in cardiomyocytes after resuscitation. The treatment inhibited autophagy activation, with lower expression levels of autophagy-associated proteins and decreased autophagosome formation in vivo and vitro. In conclusion, H2 gas inhalation after return of spontaneous circulation improved cardiac function via the inhibition of autophagy.

Multi-omics reveals a relationship between endometrial amino acid metabolism and autophagy in women with recurrent miscarriage†.

Deterioration of the endometrial environment is an essential cause of recurrent miscarriage (RM). However, current studies in terms of endometrial amino acid metabolic characterization and autophagy are still inadequate. We tried to (1) identify the alternation in metabolite profiles in the RM endometrium; (2) investigate the expression of autophagy-related proteins in RM; and (3) elucidate the association between amino acid metabolism and autophagy in RM. Our results showed that glutamine metabolites were up-regulated in the endometrium of RM women. The levels of autophagy-associated proteins, LC3B, ATG12, and Beclin-1, were significantly higher in RM. Hemostasis, autophagy and IFNα signaling were the top three differentially activated signaling pathways between women with RM and normal pregnancy. Interestingly the expression of AMPK and GCN2 was significantly up-regulated in the endometrium of women with RM, and the same expression trend was also observed in the human endometrial stromal cells cultured in glutamine deprivation medium. Furthermore, inhibition of AMPK decreased the level of GCN2, indicating a positive correlation between GCN2 and AMPK. The expression of GCN2 was consistent with the expression of ATG12 and beclin-1; however, it was opposite to that of p62. Exposure to glutamine deprivation increased the level of LC3B, GCN2, ATG12, and beclin-1. Altogether, these findings suggested significant crosstalk between amino acid metabolism and autophagy. In summary, our data suggested that aberrant crosstalk between amino acid metabolism and autophagy may contribute to the impaired endometrial microenvironment of RM. Our study may provide new insight into the diagnosis of RM due to endometrial factors.

SIRT1 suppresses cellular senescence and inflammatory cytokine release in human dermal fibroblasts by promoting the deacetylation of NF-κB and activating autophagy

Skin aging is a complex process and involves extrinsic and intrinsic processes with distinct characteristics. Understanding skin aging requires knowledge of the senescence of human dermal fibroblasts (HDFs) and the biological mechanisms involved in this process. However, the molecular mechanism responsible for the aging of HDFs is still not clear. Therefore, we investigated mechanisms of autophagy, inflammation, and cellular senescence by Western blotting, immunofluorescence, real-time PCR, and senescence-associated β-galactosidase (SA-β-gal) staining in senescent HDFs. We found SRT1720 inhibited the inductions of inflammatory cytokines and cellular senescence by deacetylating acetyl-NF-κB levels and enhancing levels of autophagy-associated proteins and SIRT1 in senescent HDFs. However, the NF-κB activator prostratin attenuated signals associated with autophagy, such as those of LC3-II and Beclin-1, but increased inflammatory cytokine levels and cellular senescence. Notably, the expression levels of SIRT1 and autophagy-associated proteins were higher in aged mice administered SRT1720 than in old mice, and SRT1720 also decreased levels of acetyl-NF-κB, inflammatory cytokines, and senescence markers, which was in accord with in vitro results. These findings support that SRT1720 acts as an anti-aging agent and inhibits the inductions of inflammatory cytokines and senescence by regulating the SIRT1/acetyl-NF-κB signaling pathway and activating autophagy in senescent HDFs.

Dexmedetomidine Protects Against Septic Liver Injury by Enhancing Autophagy Through Activation of the AMPK/SIRT1 Signaling Pathway.

Background: Liver injury is one of the serious complications of sepsis. Previous studies suggested that dexmedetomidine (DEX) could alleviate cecal ligation and puncture (CLP)-induced liver injury. However, it is unclear whether the protective effect of DEX on sepsis-induced liver injury is related to autophagy. Methods: Mice (n = 105) were randomly divided into the following groups: (i) CON group (Sham); (ii) CLP group (CLP-induced liver injury + saline); (iii) CLP + DEX group (CLP-induced liver injury + DEX). Mouse models of sepsis-induced liver injury were established using CLP. DEX or normal saline was administered by intraperitoneal injection at 0, 2, and 4 h after CLP surgery. The mortality rate within 120 h was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and inflammatory cytokines were measured at 6, 12, and 24 h in each group. Hematoxylin and eosin staining assay was carried out to detect the morphological changes of mouse liver cells in each group. The levels of autophagy-associated proteins LC3II, Beclin-1, p62, and LAMP-2 were detected in three groups of mice using western blotting. The expression of LC3II was detected using immunofluorescence. Transmission electron microscopy (TEM) of liver tissue was used to observe autophagosomes and autophagosome–lysosomes. Lastly, the effect of DEX on the AMPK/SIRT1 pathway-associated protein levels were detected using western blotting. Meanwhile, we used L0-2 cells infected with mRFP-GFP-LC3 adenovirus to further analyze the role of SIRT1 in DEX-induced autophagy in liver injury model in vitro. Results: DEX significantly improved the survival rate of septic mice at the early stage and ameliorated the pathology of sepsis-induced liver injury. The level of autophagy-associated proteins, phosphorylated (p)-AMPK/AMPK, and SIRT1 in the liver of CLP-induced sepsis mice peaked at 12 h post-CLP and decreased significantly at 24 h. In the CLP + DEX group, the levels of autophagy-associated proteins, p-AMPK/AMPK, and SIRT1 increased, whereas inflammatory cytokines decreased at 24 h. The autophagosome structure was clearly observed at different time points in the CLP + DEX group. In the in vitro hepatocyte injury model, the SIRT1 inhibitor significantly increased intracellular ROS levels and reversed the effect of DEX on autophagy flux. Conclusion: We demonstrated a novel mechanism in which DEX protects against CLP-induced liver injury. DEX enhances autophagy, which alleviates the inflammatory responses in CLP-induced liver injury by regulating the SIRT1/AMPK pathway.

The effects of UCP2 on autophagy through the AMPK signaling pathway in septic cardiomyopathy and the underlying mechanism.

BackgroundMitochondrial dysfunction plays an important role in the development of septic cardiomyopathy. This study aimed to reveal the protective role of uncoupling protein 2 (UCP2) in mitochondria through AMP-activated protein kinase (AMPK) on autophagy during septic cardiomyopathy.MethodsUCP2 knockout mice via a cecal ligation and puncture (CLP) model and the H9C2 cardiomyocyte cell line in response to lipopolysaccharide (LPS) in vitro were used to study the effect. The myocardial morphological alterations, indicators of mitochondrial injury and levels of autophagy-associated proteins (pAMPK, pmTOR, pULK1, pTSC2, Beclin-1, and LC3-I/II) were assessed. In addition, the mechanism of the interaction between UCP2 and AMPK was further studied through gain- and loss-of-function studies.ResultsCompared with the wild-type mice, the UCP2 knockout mice exhibited more severe cardiomyocyte injury after CLP, and the AMPK agonist AICAR protected against such injury. Consistent with this result, silencing UCP2 augmented the LPS-induced pathological damage and mitochondrial injury in the H9C2 cells, limited the upregulation of autophagy proteins and reduced AMPK phosphorylation. AICAR protected the cells from morphological changes and mitochondrial membrane potential loss and promoted autophagy. The silencing and overexpression of UCP2 led to correlated changes in the AMPK upstream kinases pLKB1 and CAMKK2.ConclusionsUCP2 exerts cardioprotective effects on mitochondrial dysfunction during sepsis via the action of AMPK on autophagy.

Erastin triggers autophagic death of breast cancer cells by increasing intracellular iron levels.

Erastin is a small molecular compound that induces ferroptosis by binding to voltage-dependent anion-selective channel protein (VDAC)2, VDAC3 and solute carrier family 7 member 5 inhibiting the cystine/glutamate antiporter. However, to the best of our knowledge, the mechanism of erastin-induced breast cancer cell death remains unclear. In present study aimed to explore the underlying mechanisms of the antitumor effects of erastin on breast cancer cells. Cellular viability was assessed using an MTT assay, a lactate dehydrogenase cytotoxicity assay kit was used to determine the cell death rate, the intracellular Fe2+ levels were determined using an iron colorimetric assay kit and western blotting was used to estimate the changes of autophagy-associated proteins levels. The present study demonstrated that erastin inhibited the viability of breast cancer cells and induced breast cancer cell death in a dose-dependent manner. Additionally, autophagy was activated by erastin, as demonstrated by upregulated expression levels of autophagy-associated proteins in breast cancer cells. Bafilomycin A1, 3-methyladenine and knockdown of autophagy related (ATG)5 with small interfering RNA prevented erastin-induced breast cancer cell death and inhibited the erastin-induced changes in the expression levels of the autophagy-associated proteins beclin1, ATG5, ATG12, microtubule-associated proteins 1A/1B light chain 3B (LC3B) and P62. Furthermore, erastin-induced breast cancer cell death was inhibited by an iron chelator, deferoxamine, which inhibited the increases of erastin-induced iron levels and inhibited the erastin-induced changes in the expression levels of the autophagy-related proteins beclin1, ATG5, ATG12, LC3B and P62. In summary, erastin triggered autophagic death in breast cancer cells by increasing intracellular iron levels.

Altered expression levels of autophagy-associated proteins during exercise preconditioning indicate the involvement of autophagy in cardioprotection against exercise-induced myocardial injury

Exercise has been reported to induce autophagy. We hypothesized that exercise preconditioning (EP)-related autophagy in cardiomyocytes could be attributed to intermittent ischemia–hypoxia, allowing the heart to be protected for subsequent high-intensity exercise (HE). We applied approaches, chromotrope-2R brilliant green (C-2R BG) staining and plasma cTnI levels measuring, to characterize two periods of cardioprotection after EP: early EP (EEP) and late EP (LEP). Further addressing the relationship between ischemia–hypoxia and autophagy, key proteins, Beclin1, LC3, Cathepsin D, and p62, were determined by immunohistochemical staining, western blotting, and by their adjacent slices with C-2R BG. Results indicated that exercise-induced ischemia–hypoxia is a key factor in Beclin1-dependent autophagy. High-intensity exercise was associated with the impairment of autophagy due to high levels of LC3II and unchanged levels of p62, intermittent ischemia–hypoxia by EP itself plays a key role in autophagy, which resulted in more favorable cellular effects during EEP-cardioprotection compared to LEP.

MDM2 inhibition-mediated autophagy contributes to the pro-apoptotic effect of berberine in p53-null leukemic cells

AimsBerberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. Main methodsP-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6–8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. Key findingsBBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SignificanceMDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.

Helium low temperature plasma induced HepG2 cells autophagy through ROS-mediated PI3K/AKT/mTOR/P70s6k signaling pathway

Low temperature plasma (LTP) has emerged as a new research hotspot of promising therapy to fight against cancer. Autophagy is a multifunctional process that digests and recycles cellular contents within lysosomes to maintain homeostasis, which is the underlying mechanism of cancer cell death and anti-cancer treatment. This study aimed at investigating whether autophagy of HepG2 cells could be induced by LTP and revealing the related molecular mechanism. For this purpose, the atmospheric pressure plasma jet (APPJ) in helium was utilized to generate plasma-actived medium (PAM) to treat HepG2 cells in vitro. Catalase (CAT), superoxide dismutase (SOD) and 3-methyladenine (3-MA) were respectively added into the PAM as the intervention group. The cell viability, formation of autophagosomes, intracellular reactive oxygen species (ROS) level, expression levels of autophagy-associated proteins and key proteins involved in PI3K/AKT/mTOR pathways were detected. The results showed that LTP inhibited cell viability in a dose- and time-dependent manner. Autophagy was induced through the formation of autophagosomes, conversion of LC3-II/LC3-I, increased expression of beclin 1 and degradation of p62. The mechanism was deduced that LTP enhanced the intracellular ROS level and decreased the phosphorylation level of key proteins in PI3K/AKT/mTOR/p70S6K pathway. However, these effects were blocked by the autophagy inhibitor 3-MA and ROS scavengers (CAT and SOD). The therapeutic efficacy of LTP against HepG2 cells may involve autophagy via suppressing PI3K/AKT/mTOR signaling pathway by LTP-mediated ROS.

RSL3 induced autophagic death in glioma cells via causing glycolysis dysfunction

RSL3 is a type of small molecular compound which can inactivate glutathione peroxidase 4 (GPX4) and induce ferroptosis, but its role in glioma cell death remains unclear. In this study, we found RSL3 inhibited the viabilities of glioma cells and induced glioma cell death in a dose-dependent manner. In vitro studies revealed that RSL3-induced cell death was accompanied with the changes of autophagy-associated protein levels and was alleviated by pretreatment of 3-Methyladenine, bafilomycin A1 and knockdown of ATG5 with siRNA. The ATP and pyruvate content as well as the protein levels of HKII, PFKP, PKM2 were decreased in cells treated by RSL3, indicating that RSL3 induced glycolysis dysfunction in glioma cells. Moreover, supplement of exterior sodium pyruvate, which was a final product of glycolysis, not only inhibited the changes of autophagy-associated protein levels caused by RSL3, but also prevented RSL3-induced cell death. In vivo data suggested that the inhibitory effect of RSL3 on the growth of glioma cells was associated with glycolysis dysfunction and autophagy activation. Taken together, RSL3 induced autophagic cell death in glioma cells via causing glycolysis dysfunction.

Parthenolide Inhibits the Proliferation of MDA-T32 Papillary Thyroid Carcinoma Cells in Vitro and in Mouse Tumor Xenografts and Activates Autophagy and Apoptosis by Downregulation of the Mammalian Target of Rapamycin (mTOR)/PI3K/AKT Signaling Pathway.

BackgroundThis study aimed to examine the effects of the sesquiterpene lactone, parthenolide, on migration, autophagy, and apoptosis of MDA-T32 human papillary thyroid carcinoma cells in vitro and in mouse tumor xenografts.Material/MethodsCell proliferation and viability of MDA-T32 human papillary thyroid carcinoma cells were determined by MTT assay, and cell migration was studied using a transwell assay. Fluorescence microscopy using acridine orange (AO) and ethidium bromide (EB) staining evaluated apoptosis. Transmission electron microscopy was used to study the effects of parthenolide on autophagy, and Western blot examined the levels of autophagy-associated proteins, including Bax, Bcl-2, and LC3-ll. Mice (n=10) were injected with 5×106 MDA-T32 cells subcutaneously into the left flank, and xenograft tumors were grown for six weeks. Control untreated mice (n=5) were compared with treated mice (n=5) given parthenolide three times per week.ResultsParthenolide resulted in a dose-dependent reduction in viability and cell migration of MDA-T32 cells, with a half-maximal inhibitory concentration (IC50) of 12 μM. AO and EB staining showed that parthenolide induced cell apoptosis and electron microscopy identified autophagosomes in MDA-T32 cells. Parthenolide induced increased expression of the autophagocytic proteins, LC3-II and beclin-1, had a dose-dependent inhibitory effect on the mTOR/PI3K/AKT cascade in MDA-T32 cells and inhibited the growth of the mouse xenograft tumors in vivo.ConclusionsParthenolide inhibited the growth and migration of MDA-T32 human papillary thyroid carcinoma cells in vitro and mouse tumor xenografts and activated autophagy and apoptosis by downregulation of the mTOR/PI3K/AKT signaling pathway.

Association of exercise-induced autophagy upregulation and apoptosis suppression with neuroprotection against pharmacologically induced Parkinson's disease.

[Purpose]We investigated whether treadmill exercise (TE)-induced neuroprotection was associated with enhanced autophagy and reduced apoptosis in a mouse model of pharmacologically induced Parkinson's disease (PD).[Methods]PD was induced via the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). C57BL/6 male mice were randomly assigned to the following three groups: control (C57BL, n=10), MPTP with probenecid (MPTP/C, n=10), and MPTP/ C plus exercise (MPTP-TE, n=10). The MPTP-TE mice performed TE training (10 m/min, 60 min/day, 5 days/week) for 8 weeks. The rotarod test was used to assess motor function.[Results]TE restored MPTP/P-induced motor dysfunctionand increased tyrosine hydroxylase levels. Furthermore, TE diminished the levels of α-synuclein (α-syn), a neurotoxin; modulated the levels of autophagy-associated proteins, including microtubule-associated protein 1 light chain 3-II, p62, BECLIN1, BNIP3, and lysosomal-associated membrane protein-2, which enhanced autophagy; inhibited the activation of proapoptotic proteins (caspase-3 and BAX);and upregulated BCL-2, an antiapoptosis protein.[Conclusion]Taken together, these results suggested that the TE-induced neuroprotection against MPTP-induced cell death was associated with enhanced autophagy and neuronal regeneration based on the findings of inhibited proapoptotic events in the brains of the TE-trained animals.

Increased expression of sterol regulatory element binding protein‑2 alleviates autophagic dysfunction in NAFLD.

Sterol regulatory element binding protein-2 (SREBP-2) is an important transcription factor in lipid homeostasis. A previous study showed that SREBP-2 also activated autophagic genes during cell-sterol depletion. Alterations in autophagy are reported to be involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, whether the regulation of SREBP-2 restores dysfunctional autophagy in hepatocytes during NAFLD remains to be elucidated. In the present study, a steatosis model was established with palmitic acid (PA) treatment at the indicated times and concentrations. Autophagosomes in hepatocytes were visualized by confocal microscopy after transfection with a tandem GFP-mCherry-LC3 construct. Autophagy-associated protein levels were analyzed by western blot analysis. Loss- and gain-of-function studies were performed to examine the role of SREBP-2 in the regulation of hepatocyte autophagy. It was demonstrated that PA induced autophagy and enhanced autophagic flux at the early stage, whereas prolonged treatment with PA resulted in dysfunction of autophagy in the PA-induced steatotic hepatocytes. In addition, different cellular models presented with differing dysfunctional autophagy in response to fatty acid overload. It was also confirmed that SREBP-2 regulated autophagy-related gene expression in hepatocytes, and it was shown that the overexpression of SREBP-2 increased the expression of autophagy-related genes, but did not affect the inhibition of the autophagic flux in lipid-overloaded HL-7702 cells. By contrast, increased SREBP-2 partly restored the inhibited autophagic activity in lipid-overloaded hepatoma HepG2 cells. Taken together, the present study demonstrated that autophagic function was impaired in lipid-overloaded human hepatocytes, and the differential effect of PA on autophagy was associated with the duration of PA and the cell type. Under these conditions, the overexpression of SREBP-2 alleviated the inhibited autophagic activity rather than the inhibition of autophagic flux. Consequently, the results indicated that restoration of autophagy dysfunction via the regulation of SREBP-2 may be a potential therapeutic target for the treatment of NAFLD.